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Oxidation of methionine residues in proteins to methionine sulfoxide can be reversed by the enzyme peptide methionine sulfoxide reductase (MsrA, EC 1.8.4.6). We cloned the gene encoding a human homologue (hMsrA) of the enzyme, which has an 88% amino acid sequence identity to the bovine version (bMsrA). With dot blot analyses based on RNA from human tissues, expression of hMsrA was found in all tissues tested, with highest mRNA levels in adult kidney and cerebellum, followed by liver, heart ventricles, bone marrow and hippocampus. In fetal tissue, expression was highest in the liver. No expression of hmsrA was detected in leukemia and lymphoma cell lines. To test if hMsrA is functional in cells, we assayed its effect on the inactivation time course of the A-type potassium channel ShC/B since this channel property strongly depends on the oxidative state of a methionine residue in the N-terminal part of the polypeptide. Co-expression of ShC/B and hMsrA in Xenopus oocytes significantly accelerated inactivation, showing that the cloned enzyme is functional in an in vivo assay system. Furthermore, the activity of a purified glutathione-S-transferase-hMsrA fusion protein was demonstrated in vitro by measuring the reduction of [3H]N-acetyl methionine sulfoxide.

Methionine oxidation is known to alter functional properties of a transient A-type potassium channel expressed in Xenopus oocytes. We show here that nitric oxide (NO) slows down the K+ channel inactivation time course by oxidizing a critical methionine residue in the inactivation ball domain of the channel protein. We also demonstrate that the channel protein is protected from methionine oxidation by the enzyme methionine sulfoxide reductase and the antioxidant vitamin C.

Using cysteine (Cys) scanning mutagenesis of the inward rectifier K+ channel Kir2.1, we investigated its pore structure and putative conformational changes. In the background of the Kir2.1 mutant C149F which showed no sensitivity towards Cys-modifying reagents, Cys residues were introduced at 10 positions in the H5 pore region. Out of six functional mutants, T141C and F147C showed clear changes in current amplitude when Cys-modifying reagents were applied from the external side. These results suggest that the corresponding sections of the H5 pore region face to the external side which is in contrast to the results previously obtained for voltage-gated K+ (Kv) channels. Using the mutants T141C and F147C, we investigated whether or not Kir2.1 channels show state-dependent conformational changes of the pore structure. Substantial alterations of the holding potential or external K+ concentration, however, did not cause any significant change in the speed of channel modification upon application of Cys-specific reagents, suggesting that Kir2.1 channels do not undergo conformational changes, in contrast to C-type inactivating Kv channels.

The immunosuppressant cyclosporin A (CsA) reduced the functional expression of Kir2.1 potassium channels in Xenopus oocytes in a dose-dependent manner with an IC50 of 11 microM when the oocytes were incubated with CsA after RNA injection. FK506 was less effective than CsA; cyclosporin H, a non-immunosuppressive derivative of CsA, did not have a significant effect. CsA did not impair protein synthesis since other potassium channel types (Kir1.1, Kv1.1, Kv1.4) were much less sensitive to CsA. Our results suggest that the functional expression of Kir2.1 channels is facilitated by the peptidyl-prolyl isomerase cyclophilin. The observations illustrate a new role of CsA in regulation of membrane ion transport, and may provide an alternative explanation for CsA-induced side effects in clinical use.

The class III antiarrhythmic drug clofilium is known to block diverse delayed rectifier K+ channels at micromolar concentrations. In the present study we investigated the potency of clofilium and its tertiary analog LY97241 to inhibit K+ channels, encoded by the human ether-a-go-go related gene (HERG). Clofilium blocked HERG channels in a voltage-dependent fashion with an IC50 of 250 nM and 150 nM at 0 and +40 mV, respectively. LY97241 was almost 10-fold more potent (IC50 of 19 nM at +40 mV). Other cloned K+ channels which are also expressed in cardiac tissue, Kv1.1, Kv1.2, Kv1.4, Kv1.5, Kv4.2, Kir2.1, or I(Ks), were not affected by 100-fold higher concentrations. Block of HERG channels by LY97241 was voltage dependent and the rate of HERG inactivation was increased by LY97241. A rise of [K+]0 decreased both, rate of HERG inactivation and LY97241 affinity. The HERG S631A and S620T mutant channels which have a strongly reduced degree of inactivation were 7-fold and 33-fold less sensitive to LY97241 blockade, indicating that LY97241 binding is affected by HERG channel inactivation. In summary, the antiarrhythmic action of clofilium and its analog LY97241 appears to be caused by their potent, but distinct ability for blocking HERG channels.

A novel potassium channel beta-subunit (Kv beta 3) was cloned from rat brain being the third member of a Kv beta subunit gene family. It is a protein of 403 amino acid residues with a 68% amino acid sequence homology to Kv beta 1.1. Kv beta 3 is primarily expressed in rat brain having a distribution distinct to those of Kv beta 1.1 and Kv beta 2. This subunit also has a long N-terminal structure and induces inactivation in N-terminal deleted Kv1.4 but not in other members of the Kv1 channel family. Similarly to Kv beta 1.1, the Kv beta 3-induced inactivation is regulated by the intracellular redox potential.

The SS2 and adjacent regions of the 4 internal repeats of sodium channel II were subjected to single mutations involving, mainly, charged amino acid residues. These sodium channel mutants, expressed in Xenopus oocytes by microinjection of cDNA-derived mRNAs, were tested for sensitivity to tetrodotoxin and saxitoxin and for single-channel conductance. The results obtained show that mutations involving 2 clusters of predominantly negatively charged residues, located at equivalent positions in the SS2 segment of the 4 repeats, strongly reduce toxin sensitivity, whereas mutations of adjacent residues exert much smaller or no effects. This suggests that the 2 clusters of residues, probably forming ring structures, take part in the extracellular mouth and/or the pore wall of the sodium channel. This view is further supported by our finding that all mutations reducing net negative charge in these amino acid clusters cause a marked decrease in single-channel conductance.